Cloning and Expression Characteristics Analysis of Millet Genes PmDEP1 and PmEP3

doi:10.13560/j.cnki.biotech.bull.1985.2024-0689

Abstract

Abstract:

Objective Panicle type is an important factor affecting crop yield and mechanized harvesting. DEP1 (Dense Erect Panicles 1) and EP3 (Erect Panicles 3) genes are key ones controlling spike type formation. This study aims to explore the structure and expression characteristics of PmDEP1 and PmEP3 ones in proso millet (Panicum miliaceum L.). Method PmDEP1 and PmEP3 genes were cloned and subjected to bioinformatics analysis using the scattered ear type millet varieties, broomstick millet (ZM), dense ear type millet M278, and lateral ear type millet M350 as materials. RT-qPCR was used to detect the expression patterns of PmDEP1 and PmEP3 genes in different ear types of proso millet. Result Sequence result analysis shows that, the full-length cDNA of PmDEP1 is 1 044 bp, encoding 347 amino acids. The protein domain is predicted to be PAT1. The secondary structure is dominated by random coils. The tertiary structure is highly similar with that of rice DEP1 protein. It is predicted to be located in the nucleus and has high homology with switchgrass. It has 28 phosphorylation sites and does not have transmembrane structure and signal peptide. The full-length cDNA of PmEP3 is 1 215 bp, encoding 358 amino acids. The encoded protein belongs to the F-box family. The secondary structure is dominated by random coils and extended chains. The tertiary structure is highly similar with that of rice Os02g0260200 protein. It is predicted to be located in the cytoplasm. It has high homology with switchgrass, does not contain signal peptides, has 32 phosphorylation sites, and has a small amount of transmembrane structure. The RT-PCR results shows that PmDEP1 and PmEP3 genes are expressed in different parts of the plant at different stages. PmDEP1 gene is mainly expressed in the roots at the jointing stage, with the highest expression in the leaves of broomstick millet and M278 at the heading stage, and in the stems of M350. The expressions of PmEP3 gene is the highest in the leaves at the jointing stage, in the leaves of broomstick millet and M278 at the heading stage, and in the ears of M350. Conclusion PmDEP1 and PmEP3 genes are spike type genes that may be involved in regulating the formation of proso millet spike types.

Key words: Panicum miliaceum L., spike type gene, PmDEP1, PmEP3, gene cloning, expression analysis

XU Yuan-meng, MAO Jiao, WANG Meng-yao, WANG Shu, REN Jiang-ling, LIU Yu-han, LIU Si-chen, QIAO Zhi-jun, WANG Rui-yun, CAO Xiao-ning. Cloning and Expression Characteristics Analysis of Millet Genes PmDEP1 and PmEP3[J]. Biotechnology Bulletin, 2025, 41(2): 150-162.

Figures/Tables 13

Table 1 Primer sequences for PmDEP1 and PmEP3

引物 Primer	引物序列 Primer sequence （5′-3′）	功能 Primer function
actin	TCTGTTTTTCTCTATGTGCAGGA	基因表达分析 Analysis of gene expression
PmDEP1-Q	TGCTGCTTCCATTGTTCTTGCTG
PmEP3-Q	GAGCCTCCGCAAGTGGTATGG
PmDEP1	AGTGAATTCGAGCTCGGTACATGGGGGAGGAGGTGGCG	基因克隆 Gene cloning
PmEP3	AGTGAATTCGAGCTCGGTACATGGGGTCGGAAGAGTGGGAG	基因克隆 Gene cloning

Fig. 1 Cloning and structural analysis of PmDEP1 and PmEP3 genesA: Gel electrophoresis map of PmDEP1 gene. B: Gel electrophoresis map of PmEP3 gene. C: PmDEP1 gene structure. D: PmEP3 gene structure

Fig. 2 Nucleotide and amino acid sequences and gene domains of PmDEP1 and PmEP3A: Nucleotide and amino acid sequence of PmDEP1. B: Nucleotide and amino acid sequence of PmEP3. C: PmDEP1 gene domain. D: PmEP3 gene domain

Fig. 3 Amino acid composition of the protein encoded by PmDEP1 and PmEP3

Fig. 4 Prediction of hydrophobicity/hydrophilicity and phosphorylation of PmDEP1 and PmEP3 proteinsA: Hydrophobic/hydrophilic prediction of PmDEP1 protein. B: Phosphorylation prediction of PmDEP1 protein. C: Hydrophobic/hydrophilic prediction of PmEP3 protein. D: Phosphorylation prediction of PmEP3 protein

Fig. 5 Phylogenetic tree of PmDEP1 （A） and PmEP3 （B） proteins in broomcorn millet

Fig. 6 PmDEP1 protein （A） and PmEP3 （B） protein aligned with homologous sequences of other species

Fig. 7 Prediction of secondary and tertiary structures of PmDEP1 and PmEP3 proteinsA: Secondary structure of PmDEP1 protein. B: Secondary structure of PmEP3 protein. C: Tertiary structure of PmDEP1 protein. D: Tertiary structure of PmEP3 protein

Fig. 8 Signal peptide and transmembrane domain prediction of PmDEP1 protein and PmEP3 proteinA: Prediction of PmDEP1 protein signal peptide. B: Prediction of PmEP3 protein signal peptide. C: Prediction of PmDEP1 protein transmembrane domain. D: Prediction of PmEP3 transmembrane domain

Table 2 Prediction results of subcellular localizations for PmDEP1 and PmEP3 proteins

亚细胞位置 Subcellular location	可能性 Possibility/%
亚细胞位置 Subcellular location	PmDEP1	PmEP3
细胞核	78.3	26.1
细胞质	8.7	47.8
质膜	4.3	-
过氧化物酶体	4.3	-
线粒体	4.3	26.1

Fig. 9 Cis-acting elements of PmDEP1 （A） and PmEP3 （B） promoters

Table 3 Analysis of cis-acting elements of PmDEP1 and PmEP3 promoters

基因名称 Gene name	作用元件 Functional component	核心序列 Core sequence	数量 Number	功能 Function
PmDEP1	CGTCA-motif	CGTCA	1	MeJA作用调控元件
	TGACG-motif	TGACG	1	MeJA作用调控元件
	G-box	CACGAC	4	光响应应答元件
	chs-CMA2a	TCACTTGA	1	光响应应答元件
	TCA-element	CCATCTTTTT	1	水杨酸作用元件
	P-box	CCTTTTG	1	赤霉素反应元件
	ARE	AAACCA	1	厌氧诱导调控元件
	GC-motif	CCCCCG	2	参与缺氧特异性诱导元件
	MBS	CAACTG	1	干旱诱导元件
	CCGTCC-box	CCGTCC	1	与分生组织特异性激活调节元件
	STRE	AGGGG	4	应激反应元件
PmEP3	CGTCA-motif	CGTCA	1	MeJA作用调控元件
	TGACG-motif	TGACG	1	MeJA作用调控元件
	ATCT-motif	AATCTAATCC	1	光响应应答元件
	TCT-motif	TCTTAC	2	光响应应答元件
	MRE	AACCTAA	1	MYB参与光反应结合位点
	GARE-motif	TCTGTTG	1	赤霉素反应元件
	TC-rich repeats	ATTCTCTAAC	1	赤霉素反应元件
	AuxRR-core	GGTCCAT	1	生长素反应调控元件
	AAGAA-motif	GAAAGAA	1	脱落酸反应性相关性元件
	ARE	AAACCA	2	厌氧诱导调控元件
	MBS	CAACTG	1	干旱诱导元件
	CAT-box	GCCACT	1	分生组织表达调控元件
	GCN4_motif	TGAGTCA	1	参与胚乳表达的顺式调控元件

Fig. 10 Expressions of PmDEP1 and PmEP3 genes in different parts of ZM， M350 and M278 at jointing and heading stagesA: Expressions of PmDEP1 in different parts at jointing stage. B: Expressions of PmDEP1 in different parts at heading stage. C: Expressions of PmEP3 in different parts at jointing stage. D: Expressions of PmEP3 in various parts at heading stage. Different lowercase letters indicate that the expression was significantly different at the P<0.05 level

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